This project focuses on two serine threonine protein kinases, GCK and GCKR and a tyrosine kinase PYK2. GCK and GCKR belong to a subfamily of protein kinases, which also includes GLK, HPK1, and NIK. They are characterized by an N-terminal kinase domain related to the yeast STE20 protein kinase, a large C-terminal regulatory domain, and the ability to activate the stress- activated protein kinase (SAPK, also referred to as Jun kinase or JNK) pathway. This pathway is activated by many cellular stresses, the inflammatory cytokine tumor necrosis factor (TNF) and CD40 ligand (CD40L). CD40L is present on activated T lymphocytes and interacts with its counter-receptor CD40, which is expressed by many of the important cells in the immune system, including dendritic cells, monocytes, and B- lymphocytes. Previously, we showed that GCK and GCKR are major intermediaries in CD40 and TNF-mediated SAPK activation, and that signaling through these receptors leads to the formation of a trimolecular complex between GCKR, TRAF2, and TANK. CD40 and TNFa-induced activation of germinal center kinases and the subsequent activation of stress activated protein kinases requires TRAF2. While the TRAF2 TRAF domain binds GCK and GCKR, the RING finger domain is needed for their activation. We have found that GCKR activates the SAPK pathway in a manner that depends upon TRAF2 and Ubc13, a member along with Uev1A of a dimeric ubiquitin-conjugating enzyme complex. Interference with Ubc13 function or expression inhibits both TNFa- and TRAF2-mediated GCKR and SAPK activation. TNFa signaling leads to TRAF2 polyubiquitination, TRAF2 oligomerization, and the recruitment, ubiquitination, and activation of GCKR, all sensitive to disruption of Ubc13 function. These results indicate that the assembly of a TRAF2 lysine-63 linked polyubiquitin chain by Ubc13/Uev1A is required for TNFa-mediated GCKR and SAPK activation. To better understand the role of GCK and GCKR in vivo, the murine GCK and GCKR gene have been isolated. Both genes are complicated spanning nearly 100 kilobases with more than 30 exons. This information has been used to create gene targeting constructs. Both GCK-/- and GCKR -/- mice have been created. Neither mutation dramatically alters mouse development as GCK and GCKR deficient mice are born with a normal Mendelian frequency. Studies are in progress to characterize the effects of loss of GCK or GCKR on immune cell function, hematopoiesis, and signaling through TNF related receptors. Preliminary studies indicate significant abnormalities within the immmune systems of these mice. In addition, GCK-/- GCKR-/- mice have identified in crosses from interbreeding of double heterozygotic mice. These mice will also be studied. We have also initiated a project to determine whether GCK and/or GCK participate in BAFF receptor signaling. BAFF (B cell-activating factor belonging to the TNF family), a member of the TNF family of ligands, may play a role in B cell-mediated diseases. BAFF is a survival factor for peripheral B cells and overexpressed in mice, B cell number and immunoglobulin production is increased and an autoimmune-like phenotype is observed. BAFF binds three receptors: TACI, BCMA, and BR3. We have expressed a tagged version of the BR3 receptor in a mouse B cell line, which allow us to use an antibody to the tag as a ligand and hence signal through only the BR3 receptor. This approach also avoids the poor biologic activity often found with sources of recombinant BAFF. Preliminary data indicate that the antibody signals appropriately and that it results in the activation of SAPK and MAPK. Further studies will address the importance of GCK and GCKR in BR3 signaling and whether K63 linked ubiquitination is important for signaling triggered following engagement of BR3.